Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

A tRNA modifying enzyme as a tunable regulatory nexus for bacterial stress responses and virulence.

Post-transcriptional modifications can impact the stability and functionality of many different classes of RNA molecules and are an especially important aspect of tRNA regulation. It is hypothesized that cells can orchestrate rapid responses to changing environmental conditions by adjusting the specific types and levels of tRNA modifications. We uncovered strong evidence in support of this tRNA global regulation hypothesis by examining effects of the well-conserved tRNA modifying enzyme MiaA in extraintestinal pathogenic Escherichia coli (ExPEC), a major cause of urinary tract and bloodstream infections. MiaA mediates the prenylation of adenosine-37 within tRNAs that decode UNN codons, and we found it to be crucial to the fitness and virulence of ExPEC. MiaA levels shifted in response to stress via a post-transcriptional mechanism, resulting in marked changes in the amounts of fully modified MiaA substrates. Both ablation and forced overproduction of MiaA stimulated translational frameshifting and profoundly altered the ExPEC proteome, with variable effects attributable to UNN content, changes in the catalytic activity of MiaA, or availability of metabolic precursors. Cumulatively, these data indicate that balanced input from MiaA is critical for optimizing cellular responses, with MiaA acting much like a rheostat that can be used to realign global protein expression patterns.

Lysosomal iron recycling in mouse macrophages is dependent upon both LcytB and Steap3 reductases.

Iron that is stored in macrophages as ferritin can be made bioavailable by degrading ferritin in the lysosome and releasing iron back into the cytosol. Iron stored in ferritin is found as Fe3+ and must be reduced to Fe2+ before it can be exported from the lysosome. Here we report that the lysosomal reductase Cyb561a3 (LcytB) and the endosomal reductase six-transmembrane epithelial antigen of prostate 3 (Steap3) act as lysosomal ferrireductases in the mouse macrophage cell line RAW264.7 converting Fe3+ to Fe2+ for iron recycling. We determined that when lysosomes were loaded with horse cationic ferritin, reductions or loss of LcytB or Steap3 using CRISPR/Cas9-mediated knockout technology resulted in decreased lysosomal iron export. Loss of both reductases was additive in decreasing lysosomal iron export. Decreased reductase activity resulted in increased transcripts for iron acquisition proteins DMT1 and transferrin receptor 1 (Tfrc1) suggesting that cells were iron limited. We show that transcript expression of LcytB and Steap3 is decreased in macrophages exposed to Escherichia coli pathogen UTI89, which supports a role for these reductases in regulating iron availability for pathogens. We further show that loss of LcytB and Steap3 in macrophages infected with UTI89 led to increased proliferation of intracellular UTI89 suggesting that the endolysosomal system is retaining Fe3+ that can be used for proliferation of intravesicular pathogens. Together, our findings reveal an important role for both LcytB and Steap3 in macrophage iron recycling and suggest that limiting iron recycling by decreasing expression of endolysosomal reductases is an innate immune response to protect against pathogen proliferation and sepsis.

Cytosolic replication in epithelial cells fuels intestinal expansion and chronic fecal shedding of Salmonella Typhimurium.

Persistent and intermittent fecal shedding, hallmarks of Salmonella infections, are important for fecal-oral transmission. In the intestine, Salmonella enterica serovar Typhimurium (STm) actively invades intestinal epithelial cells (IECs) and survives in the Salmonella-containing vacuole (SCV) and the cell cytosol. Cytosolic STm replicate rapidly, express invasion factors, and induce extrusion of infected epithelial cells into the intestinal lumen. Here, we engineered STm that self-destruct in the cytosol (STmCytoKill), but replicates normally in the SCV, to examine the role of cytosolic STm in infection. Intestinal expansion and fecal shedding of STmCytoKill are impaired in mouse models of infection. We propose a model whereby repeated rounds of invasion, cytosolic replication, and release of invasive STm from extruded IECs fuels the high luminal density required for fecal shedding.

Population dynamics of an Escherichia coli ST131 lineage during recurrent urinary tract infection.

Recurrent urinary tract infections (rUTIs) are extremely common, with ~ 25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.

Commensal Strains of Neisseria Use DNA to Poison Their Pathogenic Rivals.

Commensal bacteria can interfere with colonization of the host by infiltrating pathogens. In this issue of Cell Host & Microbe, Kim et al. (2019) describe an intriguing mechanism of colonization resistance driven by the mismatching of methylation patterns following uptake of commensal-derived DNA by pathogenic strains of Neisseria.

Context-Dependent Requirements for FimH and Other Canonical Virulence Factors in Gut Colonization by Extraintestinal Pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) acts as a commensal within the mammalian gut but can induce pathology upon dissemination to other host environments such as the urinary tract and bloodstream. ExPEC genomes are likely shaped by evolutionary forces encountered within the gut, where the bacteria spend much of their time, provoking the question of how their extraintestinal virulence traits arose. The principle of coincidental evolution, in which a gene that evolved in one niche happens to be advantageous in another, has been used to argue that ExPEC virulence factors originated in response to selective pressures within the gut ecosystem. As a test of this hypothesis, the fitness of ExPEC mutants lacking canonical virulence factors was assessed within the intact murine gut in the absence of antibiotic treatment. We found that most of the tested factors, including cytotoxic necrotizing factor type 1 (CNF1), Usp, colibactin, flagella, and plasmid pUTI89, were dispensable for gut colonization. The deletion of genes encoding the adhesin PapG or the toxin HlyA had transient effects but did not interfere with longer-term persistence. In contrast, a mutant missing the type 1 pilus-associated adhesin FimH displayed somewhat reduced persistence within the gut. However, this phenotype varied dependent on the presence of specific competing strains and was partially attributable to aberrant flagellin expression in the absence of fimH These data indicate that FimH and other key ExPEC-associated factors are not strictly required for gut colonization, suggesting that the development of extraintestinal virulence traits is not driven solely by selective pressures within the gut.

Similarly Lethal Strains of Extraintestinal Pathogenic Escherichia coli Trigger Markedly Diverse Host Responses in a Zebrafish Model of Sepsis.

In individuals with sepsis, the infecting microbes are commonly viewed as generic inducers of inflammation while the host background is considered the primary variable affecting disease progression and outcome. To study the effects of bacterial strain differences on the maladaptive immune responses that are induced during sepsis, we employed a novel zebrafish embryo infection model using extraintestinal pathogenic Escherichia coli (ExPEC) isolates. These genetically diverse pathogens are a leading cause of sepsis and are becoming increasingly dangerous because of the rise of multidrug-resistant strains. Zebrafish infected with ExPEC isolates exhibit many of the pathophysiological features seen in septic human patients, including dysregulated inflammatory responses (cytokine storms), tachycardia, endothelial leakage, and progressive edema. However, only a limited subset of ExPEC isolates can trigger a sepsis-like state and death of the host when introduced into the bloodstream. Mirroring the situation in human patients, antibiotic therapy reduced ExPEC titers and improved host survival rates but was only effective within limited time frames that varied, depending on the infecting pathogen. Intriguingly, we find that phylogenetically distant but similarly lethal ExPEC isolates can stimulate markedly different host transcriptional responses, including disparate levels of inflammatory mediators. These differences correlate with the amounts of bacterial flagellin expression during infection, as well as differential activation of Toll-like receptor 5 by discrete flagellar serotypes. Altogether, this work establishes zebrafish as a relevant model of key aspects of human sepsis and highlights the ability of genetically distinct ExPEC isolates to induce divergent host responses independently of baseline host attributes. IMPORTANCE Sepsis is a life-threatening systemic inflammatory condition that is initiated by the presence of microorganisms in the bloodstream. In the United States, sepsis due to ExPEC and other pathogens kills well over a quarter of a million people each year and is associated with tremendous health care costs. A high degree of heterogeneity in the signs and symptomology of sepsis makes this disease notoriously difficult to effectively diagnose and manage. Here, using a zebrafish model of sepsis, we find that similarly lethal but genetically distinct ExPEC isolates can elicit notably disparate host responses. These variances are in part due to differences in the levels and types of flagellin that are expressed by the infecting ExPEC strains. A better understanding of the variable impact that bacterial factors like flagellin have on host responses during sepsis could lead to improved diagnostic and therapeutic approaches to these often deadly infections. Podcast: A podcast concerning this article is available.

Dsc orthologs are required for hypoxia adaptation, triazole drug responses, and fungal virulence in Aspergillus fumigatus.

Hypoxia is an environmental stress encountered by Aspergillus fumigatus during invasive pulmonary aspergillosis (IPA). The ability of this mold to adapt to hypoxia is important for fungal virulence and genetically regulated in part by the sterol regulatory element binding protein (SREBP) SrbA. SrbA is required for fungal growth in the murine lung and to ultimately cause lethal disease in murine models of IPA. Here we identified and partially characterized four genes (dscA, dscB, dscC, and dscD, here referred to as dscA-D) with previously unknown functions in A. fumigatus that are orthologs of the Schizosaccharomyces pombe genes dsc1, dsc2, dsc3, and dsc4 (dsc1-4), which encode a Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. A. fumigatus null dscA-D mutants displayed remarkable defects in hypoxic growth and increased susceptibility to triazole antifungal drugs. Consistent with the confirmed role of these genes in S. pombe, both ΔdscA and ΔdscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ΔdscA and ΔdscC strains revealed that these genes are critical for A. fumigatus virulence. Reintroduction of SrbA amino acids 1 to 425, encompassing the N terminus DNA binding domain, into the ΔdscA strain was able to partially restore virulence, further supporting a mechanistic link between DscA and SrbA function. Thus, we have shown for the first time the importance of a previously uncharacterized group of genes in A. fumigatus that mediate hypoxia adaptation, fungal virulence, and triazole drug susceptibility and that are likely linked to regulation of SrbA function.